WO2013044570A1

WO2013044570A1 - Detection method of medicine for curing mastitis and hyperplasia of mammary glands

Info

Publication number: WO2013044570A1
Application number: PCT/CN2011/085022
Authority: WO
Inventors: 胡小虎; 王锁刚; 张琼
Original assignee: 西安千禾药业有限责任公司
Priority date: 2011-09-30
Filing date: 2011-12-30
Publication date: 2013-04-04
Also published as: CN103033585A

Abstract

A detection method of Ru Pi Shu capsule. The capsule is comprised of Trichosanthes kirilowii maxim skin, Dandelion, Salvia Miltiorrhiza, Peony root, Rhizoma Bolbostemmatis, Bupleurum, and Rhizoma Corydalis. Wherein, the thin layer chromatography conditions include: using silica gel G thin layer plate, and using mixed solution with the volume ration of A, B of 6-12:1-5 as the developing solvent, wherein, the solvent A is selected from benzene, toluene, dichlorotoluene, chloroform and ether; the solvent B is selected from ethyl acetate, ethyl formate, acetone, ethanol and methanol.

Description

Method for detecting medicine for treating mastitis and breast hyperplasia

The invention belongs to the field of pharmaceutical technology, and particularly relates to a method for detecting drugs for treating mastitis and breast hyperplasia, in particular to a capsule for treating mastitis and hyperplasia of mammary glands, such as chylomicron. Detection method. Background technique

It is composed of melon skin, dandelion, salvia miltiorrhiza, red peony root, earthworm, bupleurum and heather. It has a synergistic effect and conforms to the principle of traditional Chinese medicine. It can achieve the goal of treating both the symptoms and the root causes. The preparation combines the understanding of the pathogenesis of mastitis and mammary gland hyperplasia by Chinese and Western medicine, absorbs the essence of classic ancient recipe, has the effect of relieving liver and relieving stagnation, dissipating phlegm and dissipating blood stasis, and is used for treating liver qi stagnation, mammary gland caused by phlegm and blood stasis. Inflammation, breast hyperplasia. In the clinical application process of hospitals across the country, it has proved to have significant clinical efficacy, and won the trust of the majority of medical workers and patients.

In the existing method for detecting chylomicrons, the thin-layer chromatography (TLC) identification of Salvia miltiorrhiza, Radix Paeoniae Alba, and Fritillaria cirrhosa was carried out, and the thin layer chromatography (TLC) method for determining the composition of Corydalis was not established. In order to further control the quality of the product, it is necessary to improve the quality control method of the product, thereby further ensuring the quality and efficacy of the product. Summary of the invention

SUMMARY OF THE INVENTION The object of the present invention is to overcome the deficiencies of the prior art and to provide a method for detecting a drug for mastitis and mammary gland hyperplasia, which can effectively control the quality of a drug, thereby better meeting medical needs.

The object of the present invention is achieved by the following technical solutions.

A method for detecting a drug for treating mastitis and mammary gland hyperplasia, the method comprising detecting the Corydalis in the drug by thin layer chromatography, wherein the conditions of the thin layer chromatography comprise: using a silica gel G thin layer plate And a volume ratio of 6 to 12: 1-5, preferably 9: 2, A, B mixed solution as a developing agent, wherein the A solution is selected from the group consisting of benzene, toluene, chlorin, chloroform and diethyl ether, preferably Toluene; the B solution is selected from the group consisting of ethyl acetate, ethyl decanoate, acetone, ethanol and decyl alcohol, preferably acetone.

In the above detection method, the developing agent is a terpene-acetone having a volume ratio of 6 to 12: 1-5, preferably 9:2. In the above detection method, detecting the Corydalis in the drug by thin layer chromatography comprises the following steps:

(1) taking the drug, adding sterol, sonicating, filtering, the filtrate is passed through an alumina column, and the residue is dissolved in water, added with concentrated ammonia solution to make alkaline, extracted with diethyl ether, evaporated to dryness, and the residue is dissolved with decyl alcohol. As a test solution;

(2) taking a reference substance of tetrahydropalmatine and adding a sterol to prepare a reference solution;

(3) Pipetting each of the above solutions 2~5μ1, respectively, on the same thin layer chromatography plate, the volume ratio is 6~12: 1-5, preferably 9: 2 of benzene-acetone as a developing agent, unfolding, taking out, Dry and view.

Preferably, identifying the Corydalis in the drug by thin layer chromatography comprises the steps of:

(1) Take 3g of drug content, add 50ml of sterol, sonicate for 30 minutes, filter, filtrate the inner diameter of 1.5cm, the column height is 5cm alumina column, the effluent is evaporated to dryness, the residue is added with water 10ml to dissolve, add The concentrated ammonia solution was adjusted to be alkaline, and extracted with diethyl ether for 3 times, each time 10 ml, and the mixture was combined with diethyl ether, evaporated to dryness, and the residue was dissolved in 1 ml of decyl alcohol to dissolve as a test solution;

(2) taking the reference substance of tetrahydropalmatine, adding decyl alcohol to make a solution containing 0.5 mg per lml, as a reference solution;

(3) Pipette each of the above solutions to 2~5μ1, respectively, on the same silica gel G thin layer plate prepared with 1% w/v sodium hydroxide solution, with a volume ratio of 9: 2 toluene-acetone as the developing agent. Unfold, remove, dry, and remove in the iodine cylinder for 3 minutes. After absorbing the iodine adsorbed on the plate, place it under the ultraviolet light of 365 nm.

In the above detection method, the detection of Corydalis in the drug by thin layer chromatography comprises the following steps:

(2) taking the reference material of the Corydalis, and preparing the reference drug solution in the same manner as the test solution;

(3) taking a reference substance of tetrahydropalmatine and adding a sterol to prepare a reference solution;

(4) Pipetting each of the above solutions 2~5μ1, respectively, on the same thin layer chromatography plate, the volume ratio is 6~12: 1-5, preferably 9: 2 benzene-acetone as a developing agent, unfolding, taking out, Dry and view.

Preferably, the detection of Corydalis in the drug by thin layer chromatography comprises the following steps: (1) taking 3 g of the drug content, adding 50 ml of sterol, sonicating for 30 minutes, filtering, and the filtrate has an inner diameter of 1.5 cm, the column height For a 5 cm alumina column, the effluent was evaporated to dryness, and 10 ml of water was added to the residue to dissolve. The concentrated ammonia solution was adjusted to be alkaline, and extracted with diethyl ether for 3 times, 10 ml each time, combined. The ether solution was evaporated to dryness, and the residue was dissolved in 1 ml of decyl alcohol to dissolve as a test solution;

(2) taking the reference material of taurus lg, and preparing a reference drug solution in the same manner as the test solution;

(3) taking the reference substance of tetrahydropalmatine, adding decyl alcohol to make a solution containing 0.5 mg per lml, as a reference solution;

(4) Pipetting each of the above solutions to 2 ~ 5μ1, respectively, on the same silica gel G thin layer plate prepared with 1% w / v sodium hydroxide solution, with a volume ratio of 9: 2 terpene-acetone as a developing agent, Unfold, remove, dry, and remove in the iodine cylinder for 3 minutes. After absorbing the iodine adsorbed on the plate, place it under the ultraviolet light of 365 nm.

In the above detection method, the medicine may be chylomicron, which consists of melon skin, Pugongying, salvia, red peony, soil motherfish, Bupleurum, and Corydalis.

400-500 parts by weight of melon skin, 400-500 parts by weight of dandelion, 200-250 parts by weight of Salvia miltiorrhiza, 200-250 parts by weight of red peony, 100-180 parts by weight of Fritillaria, 100-150 parts by weight of Bupleurum, Corydalis 100 -150 parts by weight;

A preferred composition is as follows: 450 parts by weight of melon skin, 450 parts by weight of dandelion, 225 parts by weight of salvia miltiorrhiza, 225 parts by weight of red peony, 150 parts by weight of Fritillaria, 135 parts by weight of Bupleurum, and 135 parts by weight of Corydalis.

The formula of chylomicron is a prescription (ie, 1000 capsules). For example: melon skin 450 g, dandelion 450 g, salvia 225 g, red peony 225 g, clay frit 150 g, Bupleurum 135 g, yanhusuo 135 g.

The preparation method of the above chylomicron sputum is as follows:

The above seven flavors were taken from Yansuo and pulverized into fine powder; Salvia miltiorrhiza and Radix Paeoniae Alba were refluxed with 95% ethanol for 2 hours, and the ethanol extract was combined and filtered. The filtrate was recovered and concentrated to a relative density of 1.36 (60 ° C). The thick paste, melon skin and other five flavors were boiled three times with water, the first 2 hours, the second 1.5 hours, the third hour, combined with the decoction, filtered, and the filtrate was concentrated under reduced pressure to a relative density of 1.36. (60 ° C) thick paste, combined with the above thick paste, add Corydalis powder, stir well, vacuum dry, pulverize into a fine powder, into the plastic bottle, that is.

In a specific embodiment, the present invention provides a method for detecting a mastitis, a breast hyperplasia drug, such as the above-described chylomicron, which comprises identifying a Corydalis in the drug by thin layer chromatography, wherein Identification of Corydalis in the drug by thin layer chromatography involves the following steps:

Take 3g of drug content, add 50ml of sterol, sonicate for 30 minutes, filter, filtrate through alumina column (inner diameter 1.5cm, column height 5cm), effluent evaporated to dryness, add 10ml of water to dissolve, add concentrated ammonia solution To the alkaline, extract 3 times with shaking with diethyl ether, 10 ml each time, and combine with diethyl ether, evaporated to dryness, and the residue was dissolved in 1 ml of decyl alcohol to dissolve as a test solution. Another reference material of marijuana reference medicinal material lg, the same method to prepare a reference drug solution.

Then take the reference substance of tetrahydropalmatine and add decyl alcohol to make a solution containing 0.5 mg per lml as a control solution.

According to the thin layer chromatography (Chinese Pharmacopoeia 2010 edition of an Appendix VI B) test, draw the above three solutions each 2 ~ 5μ1, respectively, the same layer of silica gel G prepared with l% w / v sodium hydroxide solution On the plate, with phthalic acid-acetone (9: 2) as the developing agent, unroll, remove, dry, and remove the iodine in the iodine for about 3 minutes. After absorbing the iodine absorbed on the plate, set the ultraviolet lamp (365nm). View below.

In the above detection method, in the chromatogram of the test sample, fluorescent spots of the same color are displayed at positions corresponding to the chromatograms of the reference drug and the reference substance. The above identification method is negative and has no interference, and the sample has a good shape and can be used as a basis for distinguishing the eucalyptus in the chylorrhea.

The invention determines the detection method of the thin layer identification of Corydalis, and the methodological investigation proves that the method has high sensitivity and good repeatability, and the negative control has no interference, and the whole test process is highly operable, and can be used as an index for controlling the intrinsic quality of the product. one.

Compared with the prior art, the invention increases the thin layer chromatographic identification of Corydalis yanhus. The method is negative and has no interference, and the sample has good reproducibility, and can be used as a basis for distinguishing the eucalyptus in the chyle.

The invention can effectively control the quality of the chylomicron sputum by controlling the various components in the treatment of mastitis and mammary gland hyperplasia drugs, such as chylorrhea sputum, so that the quality of the chyle sputum sputum is stable and controllable, thereby Goodly meet the needs of medical care. DRAWINGS

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

Figure 1 is a thin-layer chromatographic identification diagram of Corydalis citrifolia in a mortar, wherein 1 indicates a Corydalis-negative sample; 2-4 indicates three batches of the test article; 5 indicates a Corydalis control, a medicinal material; and 6 indicates a tetrahydropalmatine reference substance;

Fig. 2 is a thin-layer chromatographic identification diagram of Corydalis citrifolia in a sputum, wherein 1 indicates a Corydalis-negative sample; 2-4 indicates three batches of the test article; 5 indicates a Corydalis control, a medicinal material; and 6 indicates a tetrahydropalmatine reference substance. The best way to implement the invention

The invention is illustrated by the following examples, which are intended to be illustrative of the invention and are not intended to limit the scope of the invention. Example 1 Formulation and preparation method of chylomicron

Prescription: 450 g of melon skin, 450 g of dandelion, 225 g of salvia miltiorrhiza, 225 g of red peony, 150 g of Fritillaria, 135 g of Bupleurum, 135 g of Corydalis. The preparation method of the above chylomicron sputum is as follows:

The above seven flavors were taken from Yansuo and pulverized into fine powder; Salvia miltiorrhiza and Radix Paeoniae Alba were refluxed with 95% ethanol for 2 hours, and the ethanol extract was combined and filtered. The filtrate was recovered and concentrated to a relative density of 1.36 (60 ° C). The thick paste, melon skin and other five flavors were boiled three times with water, the first 2 hours, the second 1.5 hours, the third hour, combined with the decoction, filtered, and the filtrate was concentrated under reduced pressure to a relative density of 1.36. (60 ° C) thick paste, combined with the above thick paste, add Corydalis powder, stir well, vacuum dry, pulverize into a fine powder, into the plastic bottle, that is. Example 2 Identification of Corydalis yanxis by TLC

1. Test conditions: Double-slot expansion cylinder (Shanghai Xinyi); Silica gel G (for thin layer chromatography, Qingdao Ocean Chemical Plant); Carboxymethyl cellulose sodium (National Pharmaceutical Group Chemical Reagent Co., Ltd.); Thin-layer plate homemade, thickness 0.3mm. The remaining reagents were of analytical grade.

2, test steps: Take 20110513, 20110514, 20110615 three different batches of chylomicrons (purchased from Xi'an Qianhe Pharmaceutical Co., Ltd., its composition, ratio and preparation method as shown in Example 1) 3g, 50ml of sterol, sonicated for 30 minutes, filtered, the filtrate passed through an alumina column (inner diameter 1.5cm, column height 5cm), the effluent was evaporated to dryness, the residue was added with water 10ml to dissolve, and the concentrated ammonia solution was adjusted to alkaline. The mixture was extracted three times with diethyl ether, 10 ml each time, and the mixture was combined with diethyl ether, evaporated to dryness, and the residue was dissolved in 1 ml of decyl alcohol to dissolve as a test solution.

Take the reference material of Corydalis lg, and prepare the reference drug solution in the same way.

Then take the negative sample containing Corydalis, which is prepared according to the prescription ratio of the chylomicron and the process, and prepare a negative control solution with the preparation method of the test solution.

According to the thin layer chromatography (Chinese Pharmacopoeia 2010 edition, an appendix VI B) test, take 2 ~ 5μ1 of the above solution, respectively, on the same silica gel G thin layer plate prepared with 1% w / v sodium hydroxide solution, Benzene-acetone (9: 2) is a developing agent, unrolled, taken out, dried, and taken out in an iodine cylinder for about 3 minutes. After absorbing the iodine adsorbed on the plate, it is examined by ultraviolet light (365 nm). As shown in Figure 1.

The results showed that in the chromatograms of the three batches of test samples, fluorescent spots of the same color were observed at the positions corresponding to the chromatogram of the reference drug. In the chromatogram of the negative sample, there is no fluorescent spot of the same color at the corresponding position of the chromatogram of the reference drug, and the negative is non-interference. It is indicated that the other medicinal tastes in the prescription do not affect the detection of Corydalis yanhusuo. Therefore, it can be used as a basis for the identification of Corydalis in the sputum. Example 3 Identification of Corydalis yanxis by TLC

1. Test conditions: Double-slot expansion cylinder (Shanghai Xinyi); Silica gel G (for thin-layer chromatography, blue Island Marine Chemical Plant); Carboxymethyl Cellulose Sodium (National Pharmaceutical Group Chemical Reagent Co., Ltd.); Self-made thin layer plate, thickness 0.3mm. The remaining reagents were of analytical grade.

2. Test procedure: Take chylomicron (purchased from Xi'an Qianhe Pharmaceutical Co., Ltd.) 3g of content, add 50ml of sterol, sonicate for 30 minutes, filter, and filter the alumina column (inner diameter 1.5cm, Column height 5cm), the effluent is evaporated to dryness, the residue is added with water 10ml to dissolve, and the concentrated ammonia solution is added to alkaline. It is extracted with ether for 3 times, 10ml each time, combined with ether solution, evaporated to dryness, and the residue is added with 1ml of sterol. Dissolved as a test solution.

Another reference substance of tetrahydropalmatine was added, and a solution containing 0.5 mg per ml was prepared by adding decyl alcohol as a control solution.

According to the thin layer chromatography (Chinese Pharmacopoeia 2010 edition, an appendix VI B) test, draw 2μ1 of the above solution, respectively, on the same silica gel G thin layer plate prepared with 1% w/v sodium hydroxide solution, with benzene - Acetone (6: 1) is a developing agent, unrolled, taken out, dried, and taken out in an iodine cylinder for about 3 minutes. After the iodine adsorbed on the plate is exhausted, it is examined by ultraviolet light (365 nm).

The results showed that in the chromatogram of the test sample, fluorescent spots of the same color were observed at the positions corresponding to the chromatogram of the reference drug. In the negative sample chromatogram, there is no fluorescent spot of the same color at the corresponding position of the chromatogram of the reference drug, and the negative is non-interfering. It is indicated that the other medicinal tastes in the prescription do not affect the detection of Corydalis yanhusuo. Therefore, it can be used as a basis for the identification of Corydalis in the sputum. Example 4 Identification of Corydalis yanxis by TLC

Another reference substance of tetrahydropalmatine was added, and a solution containing 0.5 mg per lml was prepared by adding decyl alcohol as a control solution.

The negative sample containing no Corydalis was prepared according to the prescription ratio and the preparation method of the sputum sputum sputum sputum, and the negative control solution was prepared by the method for preparing the test solution. According to the thin layer chromatography (Chinese Pharmacopoeia 2010 edition of an Appendix VI B) test, the above solution was taken 5μ1, respectively, on the same silica gel G thin layer plate prepared with 1% w/v sodium hydroxide solution, with benzene - Acetone (12: 5) is a developing agent, unrolled, taken out, dried, and taken out in an iodine cylinder for about 3 minutes. After the iodine adsorbed on the plate is exhausted, it is examined by ultraviolet light (365 nm).

The results showed that fluorescent spots of the same color were displayed at positions corresponding to the chromatogram of the control. In the chromatogram of the negative sample, there is no fluorescent spot of the same color at the position corresponding to the chromatogram of the reference substance, and the negative is non-interfering. It is indicated that the other medicinal tastes in the prescription do not affect the detection of Corydalis, therefore, it can be used as a basis for the identification of Corydalis in the sputum. Example 5 Identification of Corydalis chinensis by TLC (Controlled Trial)

2. Test procedure: Take chylomicron (purchased from Xi'an Qianhe Pharmaceutical Co., Ltd.) 3g of content, add 50ml of sterol, sonicate for 30 minutes, filter, filtrate through alumina column (inner diameter

1.5cm, column height 5cm), the effluent is evaporated to dryness, the residue is added with water 10ml to dissolve, and the concentrated ammonia solution is adjusted to alkaline. It is extracted with ether for 3 times, 10ml each time, combined with ether solution, evaporated to dryness, and the residue is added with hydrazine. The alcohol was dissolved in 1 ml as a test solution.

According to the thin layer chromatography (Chinese Pharmacopoeia 2010 edition of an Appendix VI B) test, the above solution was taken 5μ1, respectively, on the same silica gel G thin layer plate prepared with 1% w/v sodium hydroxide solution, with benzene - Acetone (15: 0.5) is a developing agent, unrolled, taken out, dried, and taken out in an iodine cylinder for about 3 minutes. After absorbing the iodine adsorbed on the plate, it is examined by ultraviolet light (365 nm). The result is shown in the figure. 2 is shown.

The results showed that in the chromatogram of the test sample, there was no fluorescent spot of the same color at the position corresponding to the chromatogram of the reference drug and the reference solution, and there was no experimental feature, which was significantly different from the idea of our design. Therefore, it cannot be used as a developing agent.

Claims

Rights request

A method for detecting a drug for treating mastitis and mammary gland hyperplasia, which comprises identifying a Corydalis in the drug by thin layer chromatography, wherein the conditions of the thin layer chromatography include: using a silica gel G thin The mixture of A and B is 6~12: 1-5, preferably 9:2, as a developing agent, wherein the A solution is selected from the group consisting of benzene, toluene, dichlorobenzene, chloroform and diethyl ether. Preferably, the benzene is selected from the group consisting of ethyl acetate, ethyl decanoate, acetone, ethanol and decyl alcohol, preferably acetone.

The detecting method according to claim 1, wherein the developing agent is terpene-acetone having a volume ratio of 6 to 12: 1-5, preferably 9:2.

The detection method according to claim 1 or 2, wherein detecting the Corydalis in the drug by thin layer chromatography comprises the following steps:

(3) Pipetting each of the above solutions to 2 ~ 5μ1, respectively, on the same thin layer chromatography plate, the volume ratio of 6~12: 1~5, preferably 9: 2 of benzene-acetone as a developing agent, unfolding, taking out, Dry and view.

The detection method according to any one of claims 1 to 3, wherein detecting the Corydalis in the drug by thin layer chromatography comprises the following steps:

(2) taking the reference substance of tetrahydropalmatine, adding decyl alcohol to make a solution containing 0.5 mg per lml as a reference solution;

(3) Pipetting each of the above solutions to 2 ~ 5μ1, respectively, on the same silica gel G thin layer plate prepared with 1% w/v sodium hydroxide solution, with a volume ratio of 9: 2 toluene-acetone as a developing agent. Unfold, remove, dry, and remove in the iodine cylinder for 3 minutes. After absorbing the iodine adsorbed on the plate, place it under the ultraviolet light of 365 nm.

The detection method according to any one of claims 1 to 4, wherein detecting the Corydalis in the drug by thin layer chromatography comprises the following steps:

(4) Pipetting each of the above solutions to 2 ~ 5μ1, respectively, on the same thin layer chromatography plate, the volume ratio of 6~12: 1~5, preferably 9: 2 of benzene-acetone as a developing agent, unfolding, taking out, Dry and view.

The detection method according to any one of claims 1 to 5, wherein detecting the Corydalis in the drug by thin layer chromatography comprises the following steps:

The detection method according to any one of claims 1 to 6, wherein the medicine is composed of melon skin, dandelion, salvia, red peony, soil motherfish, Bupleurum, and Corydalis, and the ratio thereof is 7 :3⁄4 mouth:

The detection method according to any one of claims 1 to 7, wherein the preparation method of the medicine is as follows:

The above seven flavors were taken from the genus Corydalis and pulverized into fine powder; the salvia miltiorrhiza and the red peony were extracted with 95% v/v ethanol twice, each time for 2 hours, combined with the ethanol extract, filtered, and the filtrate was recovered and concentrated to 60 ° C. Thick cream with a density of 1.36, the other five flavors such as melon skin and boiling water for three times, the first 2 small When the first 1.5 hours, the first 1 hour, the combined decoction, filtered, the filtrate was concentrated under reduced pressure into a thick paste with a relative density of 1.36 at 60 ° C, combined with the thick paste, added to the fumarate powder, stirred evenly , vacuum drying, pulverization into fine powder, into the plastic bottle, that is.